- Geförderte Personen
Natur- und Biowissenschaften, Medizin
Elucidation of the intra- and intermolecular electron transfer mechanism of a new cellulose degrading enzyme system
BOKU, Wien, TU Berlin
01.01.2013 - 01.01.2016
A novel bi-enzymatic system, which assists cellulases in the degradation of cellulose has been recently identified in wood degrading fungi. Two oxidoreductases are involved: (a) Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome catalyzing the oxidation of β-1,4 linked cellobiose or higher cellodextrins. This reaction takes place at the dehydrogenase domain of CDH containing FAD as prosthetic group (DHCDH). In a ubsequent, intramolecular electron transfer step, the electrons are shuttled to a second domain (CYTCDH) containing a b-type heme. (b) Glycoside hydrolase family 61 (GH61) was classified according to its endo-1,4-α-D-glucanase activity. However, it was only recently shown that the elimination of the glycosidic bonds of cellulose is initiated by a monooxygenation step. The electrons required for this polysaccharide monooxygenase activity are provided by CYTCDHThe elucidation of this intermolecular electron transfer from the DHCDH via the CYTCDH to the copper enzyme GH61 is of highest importance to optimize this novel oxidative cellulolytic enzyme system for applications in the biofuel industry. The CDH/GH61 system has the capability to revolutionize the production of second generation biofuels from cellulosic energy plants. Increased efficiency and acceleration of the enzymatic hydrolysis process will boost the production of biofuels from non-food plants and result in a cost reduction of e.g. bioethanol. The fundamentals of this oxidative cellulose degradation pathway, especially the electron transfer mechanism, have to be fully understood before applied research can develop and optimize industrial scale applications. This project focuses on the CDH and GH61 enzymes from the model organism Neurospora crassa. The main objectives are to: (i) elucidate the mechanism and regulation of this enzyme system by kinetic and thermodynamic interaction studies and (ii) study the structural paradigms of the intermolecular electron transfer. Heterologous expression of all enzymes has already been established in the yeast Pichia pastoris, which will serve as molecular engineering and expression platform. Engineering of the protein interfaces by site-directed mutagenesis and domain switching will be combined with state-of-theart analytical techniques to perform fast kinetic, electrochemical and calorimetric studies in solution. During six months of the thesis project, surface enhanced Raman spectroscopy will be performed in the renowned group of Peter Hildebrand at the Technical University of Berlin to study the intermolecular electron transfer on surfaces.